The need for endodontic therapy in the future will exceed today's annual 27 million treatments. Deep caries and iatrogenic or physical trauma can destroy enamel and dentin that surrounds and protects the pulp. Conservative pulpal therapy is aimed at preventing infection, reducing inflammation, and stimulating self repair by the pulp. In response to irritation, reparative dentin (RPD) is secreted to create a protective barrier in the pulp cavity. This project is designed to elucidate the mechanisms of stimulation and control of reparative dentin formation (RPD) in the human tooth with the goal of improving the rate of natural pulpal repair. The specific aims are: 1) to establish a system of organ culture in the tooth crown or in intact segments of isolated peripheral pulp and to induce RPD by growing the above cultures in media and/or on substrates containing known promoters of mineralized tissue formation; 2) to establish cultures of isolated pulp cells in order to seed growing cultures with new and potentially competent dentin-secreting cells or to study directly the formation of RPD by these cells; 3) to explore the mechanism of action of calcium hydroxide by extracting freshly procured root dentin with calcium hydroxide, guanidinium hydrochloride/EDTA or acid and comparing the proteins or protein fragments derived from such treatments to determine if any of these substances preferentially release growth factors or noncollagenous bone proteins known to be sequestered in dentin; 4) to study the effects of growth factors upon the proliferation of pulp cells; and 5) to study the effects of calcium hydroxide, phosphoric acid, EDTA, or citric acid treatment of dentinal tubules on the viability of odontoblasts and their ability to produce RPD in vitro. The tooth crown, pulp segment, or isolated cells from transected teeth will be cultured and then examined by light microscopy and transmission and scanning electron microscopy. Cell counting, morphology, and 3H-proline labeling will be used to compare the course of matrix synthesis and mineralization in the human tooth with that of both fetal bovine pulp and cultured bone cells. Calcium content of acid solubilized samples from cultures will be measured with a calcium electrode. The activity of alkaline phosphatase will be monitored by spectrophotometric assay. Extracted dentin growth factors, identified with appropriate antibodies, will be analyzed using heparin sulfate column